The present invention relates to production of recombinant Follicle-Stimulating Hormone (FSH). FSH belongs the class of gonadotropins.
FSH is used in the treatment of infertility and reproductive disorders in both female and male patients. FSH is used in female patients in ovulation induction (OI) and in controlled ovarian hyperstimulation (COH), e.g. for assisted reproductive technologies (ART). In a typical treatment regimen for ovulation induction, a patient is administered daily injections of FSH or a derivative thereof (about 75 to 300 IU RFSH/day) for a period of from about 6 to about 12 days. In a typical treatment regimen for controlled ovarian hyperstimulation, a patient is administered daily injections of FSH or a derivative thereof (about 150-600 IU RFSH/day) for a period of from about 6 to about 12 days. FSH is also used to induce spermatogenesis in men suffering from oligospermia. A regimen using 150 IU FSH 3 times weekly in combination with 2′500 IU hCG twice weekly has been successful in achieving an improvement in sperm count in men suffering from hypogonadotrophic hypogonadism. Because of the importance of FSH in the treatment of fertility disorders, the provision of FSH of high stability and of high specific activity is desirable.
In nature, FSH is produced by the pituitary gland. For pharmaceutical use, FSH may be produced recombinantly (rFSH), or it may be isolated from the urine of postmenopausal females (uFSH). The manufacturing process of rFSH necessitates two main steps: cultivation of a genetically engineered cell expressing FSH, and purification of the protein. The protein is then formulated with a pharmaceutically acceptable carrier in order to obtain a pharmaceutical composition.
For culturing cells, in the past the culture medium used to be supplemented with serum, which served as a universal nutrient for the growth and maintenance of all mammalian cell lines. However, the advent of BSE (Bovine Spongiform Encephalopathy), a transmissible neurodegenerative disease of cattle with a long latency or incubation period, has raised regulatory concerns about using animal-derived sera in the production of biologically active products. Therefore, it is currently preferred to produce recombinant proteins using serum-free media. Such media are well known in the art and commercialized by several companies such as, e.g., Sigma, BioWhittaker, Gibco BRL, Cambrex and JRH.
One of the problems encountered when storing rFSH is the presence of oxidized forms of FSH. To partially solve this problem, an antioxidant may be added to the pharmaceutical composition in order to stabilize the FSH protein during storage before administration to the patient in need of treatment. For example, EP 0 853 945 (Skrabanja and Van den Oetelaar, 1998) describes liquid gonadotropin-containing formulations in admixture with a stabilizer such as, e.g., sodium citrate at 25-100 mM and L-methionine at 1-10 mM. It is shown that such formulations allow storage of FSH formulations for longer times. WO 92/15614 (Takruri H., 1992) also relates to a method for inhibiting oxidation of a polypeptide in a liquid or a semi-liquid pharmaceutical composition such as, e.g., a storage medium or an aqueous ophthalmic solution. Specifically, WO 92/15614 shows that an ophthalmic solution or an ophthalmic ointment comprising L-methionine at a concentration of 10 mg/L stabilizes the Human Epidermal Growth Factor. However, the above documents only disclose the use of an antioxidant in a pharmaceutical formulation, and not in a culture medium.
Amino acids and compounds exhibiting antioxidant activity can also be added to culture media, either as nutrients, or for protecting cell lines for cell death. For example, U.S. Pat. No. 4,560,655 discloses a serum-free medium comprising approximately 30 mg/L of L-methionine, said medium being used for cultivation of swine testicle cells, AG14 myeloma cells and murine spleen cells. WO 95/12664 teaches a method by which the disadvantage due to insufficient amounts of various growth-limiting factors, one of them being the L-methionine amino acid, can be overcome for a particular cell line. Specifically, WO 95/12664 teaches a method for adapting the CHO E5F3G cell line, which expresses human M-CSF, to grow at increased cell density. In this method, the CHO E5F3G cells are grown in a medium comprising 104 mg/L of L-methionine (see the Example and Table 2). Yun et al. teaches that the addition of a combination of glutathione and of iron chelators into the culture medium reduces the cell death of CHO cells (Yun et al., 2003). Saito et al. further teaches that various antioxidants may be used in culture media to protect a cell line from cell death (Saito et al., 2003). However, WO 95/12664, U.S. Pat. No. 4,560,655, Yun et al. and Saito et al. are silent on the potential effect (if any) of amino acids, glutathione and iron chelators on the oxidation state of the recombinant protein produced by the cell line. In conclusion, these documents only disclose the use of L-methionine or of glutathione combined to iron chelators for improving the growth and/or the viability of the cultured cells.
WO 99/50390 relates to a culture medium for producing interferon-α from leukocytes, said culture medium comprising methionine. It is demonstrated by HPLC that the quality of the interferon-α protein after purification is improved upon addition of methionine into the culture medium. The inventors of WO 99/50390 hypothesize that this improvement may be due to decreased oxidation of the interferon-α protein. WO 99/50390 further indicates that a too low amount of methionine results in a decreased effect, and that a too high amount causes lower interferon yields. Specifically, WO 99/50390 teaches that a range of about 50 to 100 mg/L is an especially preferred range when producing interferon-α from leukocytes. In addition, WO 99/50390 only contemplates a medium for production of interferon-α, which is a monomeric protein. WO 99/50390 neither mentions nor suggests a medium for production of dimeric hormones such as, e.g., FSH, which is solely secreted upon dimerization (Matzuk et al., 1988).
In summary, none of the documents mentioned above relates to the use of an antioxidant in a serum-free culture medium for reducing the oxidation of dimeric gonadotropins.